A central problem in mammalian reproductive biology is to understand the mechanisms by which a few follicles are selected to survive atresia to ovulate their eggs into the oviduct to be fertilized. It is clear that the concentration of FSH within the microenvironment has a critical role in regulating this process, eg. relatively high and low in levels of FSH in follicular fluid are associated with selection and atresia respectively. Despite intensive studies, the mechanism by which the concentration of follicular fluid FSH controls the developmental fate of a follicle remains largely unknown. During the course of this grant, we have made several novel discoveries that suggested that the level of FSH-induced granulosa cytodifferentiation is regulated, in part, by the production of two intrinsic proteins which might act in an autocrine/paracrine manner to inhibit folliculogenesis. These discoveries include: l) two novel genes, insulin-like growth factor binding protein -4 and -5 (IGFBP-4, -5) are induced in rat granulosa cells during atresia; 2) the levels of IGFBP-4 and -5 expression are regulated in a biphasic fashion by FSH e.g. low and high doses stimulate and inhibit IGFBP expression respectively; and 3) exogenous IGFBP-4 and -5 in vitro inhibit FSH-induced estradiol and progesterone production by granulosa cells. These results support the intriguing hypothesis that IGFBP-4 and -5 might serve as inducers of atresia in vivo. The challenge is to prove this hypothesis. At present, very little is known about how IGFBP-4 and -5 act and interact to regulate FSH-induced granulosa growth and cytodifferentiation in vitro, and nothing is known about their effects on folliculogenesis in vivo. The ultimate goal of this proposal is to fill this fundamental gap in our knowledge. Three specific aims are proposed. Specific Aim #1: to express recombinant rat (rr) IGFBP-4 and -5 in CHO cells and to generate polyclonal antibodies to the mature proteins. Specific Aim #2: to use the rrIGFBPs and their polyclonal antibodies to characterize the biological effects of these novel peptides on rat granulosa cells in vitro. In serum free cell culture, cells will be incubated alone and together with FSH, rrIGFBPs and/or their antibodies. After culture, the cells will be analyzed for differentiation markers, mitosis, and apoptosis. In parallel studies, the effects of these peptides on LH and prolactin induced responses will be examined. Specific Aim #3: to determine the effects of exogenously administered rrIGFBP-4 and -5 and their antibodies on granulosa proliferation, differentiation and apoptosis in vivo. Because IGFBP-4 and -5 are present in human follicles, an understanding of how these intrinsic proteins modulate, either amplify or attenuate, specific FSH-induced granulosa responses might provide new insight into ovulation disorders in women.